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Nucleic Acids Research, 1991, Vol. 19, No. 8 1933-1939
© 1991

MOLECULAR BIOLOGY

Characterization of the promoter for the rat and human link protein gene

Craig Rhodes*, Pierre Savagner, Sergio Line, Makoto Sasaki, Mike Chirigos, Kurt Doege+ and Yoshihiko Yamada

Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health Bethesda, MD 20892, USA

* To whom correspondence should be addressed at George Washington University Genetics Program

Received October 5, 1990. Revised March 14, 1991. Accepted March 14, 1991.

We have isolated the 5'-end of the gene for the rat and human link protein by screening genomic libraries with oligonucleotides corresponding to the 5'-cDNA sequence. Several overlapping clones were isolated for the human link protein gene, while only one clone was obtained for the rat. All the clones contained a single exon of which the sequence was identical to the moat 5'-end of the rat and human cDNAs. Transcription initiation sites for the rat link gene were identified by primer extension and S1 protection analysis using total RNA from the rat Swarm chondrosarcoma. Transcriptional initiation sites for the human link gene were determined by specific primer extension of RNA from human fetal cartilage. Comparison of 1500 bp of 5'-flanking sequence between the rat and human link protein genes showed strong sequence conservation near the start site of transcription with 80% overall identity. Analysis of the 5'-flanking regions also revealed a large inverted repeat consisting of repeating purine-pyrimidine, which has the potential to form left handed Z-DNA.

Transcriptional regulation of the link protein gene was studied by coupling either 7.0 kb or 0.85 kb of 5'-flanking rat DNA to the chloramphenicol acetyltransferase (CAT) gene followed by transfection into chick embryonic chondrocytes (CEC) and HeLa cells. Both constructs had considerable CAT activity in CEC cells and less activity in HeLa cells. Furthermore, inclusion of a DNA fragment from the first intron increased relative CAT activity in both of these cell types. The increased activity from the first intron was shown to be orientation independent in CEC. These results indicate the presence of positive cia-acting regulatory elements in both the promoter and first intron of the rat gene for link protein.

+Present address: Research Unit, Shriner's Hospital for Crippled Children, Portland, OR 97201, USA


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